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Flow Cytometry General Protocol

The protocols described below are for general application. Any product specific protocol supercedes these general recommendations.

Background

Staining of cells using monoclonal antibodies is used extensively to characterize cell surface antigens when monitoring extracellular antigens. Flow Cytometry data have been used to identify the families of cell types based on surface antigens and to group antibodies based on their recognition of these antigens. The use of flow cytometry for extracellular staining continues to provide useful data for identifying and separating specific cell populations. The method requires specialized equipment in which a mixture of cells can be separated based on the presence or absence of the expected emission wavelength of a specific fluorescent conjugate.

Common Examples of Fluorescent Dyes: Peak Wavelength (nm)
FluorochromeAbbreviationAbsorptionEmission
FluoreceinFITC495530
RhodamineRhod555620
R-PhycoerythrinrPE490, 546575
Texas RedTR568590
R-Cy5Cy5488680

Flow Cytometry Protocol for Extracellular Antigens

Note: Fluorescent reagents should be protected from light.
Materials and Equipment
  • Sample cells
  • 12 x 75mm test tubes
  • PBS, 0.1% sodium azide, 1% FBS
  • Fluorescent-labeled Antibody
  • Centrifuge
  • Vortex mixer
  • Flow cytometer
  • PBS, 0.5% Paraformaldehyde, 4°C
  • Pipettes
  • PBS/ sodium azide at 4°C
Procedure
  1. Prepare the desired biological cells according to the appropriate protocol. Adjust the concentration of the cells to 2x10e7 cells per ml (or 1x10e6 cells per 50ul: each test consists of 1x10e6 cells), diluting with PBS, 0.1% sodium azide, 1% FBS or BSA, as necessary. The cells can be isolated up to 4 hours before being stained as long as they are kept on ice. Note: In some cases, use of a non-specific binding or blocking agent may be desired. If so, add the blocking agent (such as normal serum or BSA) and incubate at RT for 10 minutes prior to the addition of antibody to the cells.
  2. Dilute the fluorescent conjugated antibody appropriately according to the specific recommended dilution for the staining being done. Use PBS, 0.1% sodium azide, 1% FBS for the dilution. Dilute the antibody so that a volume of 10-20ul is added to the cells.
  3. Incubate the cells with the antibody at 4°C for 30 minutes in the dark. All conjugated antibodies (FITC, R-PE, etc.) for double or triple staining can be added simultaneously at this point and do not require additional incubations.
  4. After incubation remove the unbound antibody from the cells by washing with 1ml of PBS, 0.1% sodium azide, 1% FBS. Pipette the PBS, 0.1% sodium azide, 1% FBS into each tube, vortex, then centrifuge at 350xg for 5-7 minutes at 4°C. Carefully aspirate the supernatant leaving about 50-100ul in the bottom of each tube. Repeat this process for a total of 3 washes.
  5. If flow cytometry is to be performed the same day, resuspend the cells in 0.5ml of cold PBS, 0.1% sodium azide, 1% FBS after aspirating the supernatant. Gently vortex and analyze the cells. If analysis will not be performed the same day, the cells may be fixed in 0.5ml cold PBS with 0.5% paraformaldehyde and stored at 2-8°C in the dark in buffer containing 0.1% sodium azide for as long as 48 hours.

Flow Cytometry for Intracellular Antigens

Cell Fixation and Permeabilization

Note: Cells should be kept on ice unless otherwise specified

  1. Wash the cells twice with cold PBS.
  2. Fix the cells with 2% paraformaldehyde in cold PBS for 15 minutes (4°C). It is very important to assure that the cells are uniformly resuspended during fixation.
  3. Wash the cells twice with cold PBS.
  4. Permeabilize the cells with 100% ice-cold methanol added dropwise while the cells are gently vortexing. Again, it is vitally important that the cells are uniformly suspended. Allow the cells to soak in cold methanol for 15 minutes. (Alternate permeabilization methods include the use of 0.1% saponin or 0.1% Tween-20.)
  5. Wash the cells twice with cold PBS.
Antibody Incubation

Note: Use isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.

  1. Add approximately 1x10e6 cells to each flow cytometry tube and wash with 1 ml of 0.1% saponin or Tween 20 diluted in PBS with 2% FBS added.
  2. Resuspend cells in 1 ml of 0.1% saponin (or Tween 20) + 2% FBS and incubate for 30 minutes at RT. The purpose of this step is to block non-specific binding.
  3. Add 20ul of antibody diluted to the recommended concentration in 100 ul of the above blocking solution and incubate for 20 minutes at RT.
  4. If the primary antibody is not conjugated with a fluorochrome, then a second incubation with a fluorochrome-conjugated secondary antibody will be necessary. Incubate 20 minutes in blocking solution at RT.
  5. Wash the cells once with blocking buffer, then finally with PBS
  6. Resuspend the cells in 500ul PBS and run on flow cytometer.

Troubleshooting

Problem: No staining
  1. Make sure that the appropriate primary or secondary antibodies have been added, and that the secondary antibody has the correct host and source to correctly bind to the primary antibody.
  2. Is the secondary antibody active? Has it been used successfully with other primary antibodies?
  3. If the fluorochrome used is Phycoerythrin or Allophycocyanin based, make sure that the label has not been frozen.
  4. Is the target antigen present on test tissue? Check literature for antigen expression and incorporate a positive control of known antigen expression alongside test material.
  5. Does antibody recognize antigen in test species? Check that antibody cross-reacts with species being used. Not all antibodies will cross-react across species.
  6. Ensure that correct laser is being used to excite fluorochrome, and that correct channel is being used to analyze emissions.
Problem: Non-specific staining
  1. Non-specific staining may be due to autofluorescence. Solution: check levels of autofluorescence by including a tube of cells only (i.e. without any antibody) into your panel.
  2. Certain cells express low affinity Fc receptors, which bind whole antibodies via Fc region. Use Fc blocking agents.
  3. Non-specific staining may be due to the secondary antibody. Select a secondary antibody that will not cross-react with target tissue.
  4. Make sure that sufficient washing steps have been included.
  5. Titrate the test antibody carefully. Non-specific staining may be reduced at lower antibody concentrations.
Problem: Weak staining
  1. Weak staining may be due to overdilution of antibodies. Make sure that antibodies are used at the correct concentration by titrating antibodies before use.
  2. Weak staining may be due to an excess cell number. Adjust cell population to recommended density.
  3. Weak staining may be due to lack of antigen expression. Check literature for expected levels of expression.
  4. If antigen expression is weak, select an antibody that is conjugated to a brighter fluorochrome.
  5. Incubation time and temperature with either primary or secondary antibody should be optimized.
Problem: Unexpected staining profile
  1. Ensure that cells are used as fresh as possible. Profile may be showing dead cells and debris.
  2. Lysing solutions may affect certain antigens. Select a method that does not interfere with antigen detection.
  3. Some antigens are expressed intracellularly and, therefore, cell permeabilization methods may be required.
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